Download Progressors and Long-Term Nonprogressors T Cells in HIV

Maintenance of Large Numbers of
Virus-Specific CD8 + T Cells in HIV-Infected
Progressors and Long-Term Nonprogressors
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of June 17, 2017.
Juan C. Gea-Banacloche, Stephen A. Migueles, Lisa
Martino, W. Lesley Shupert, Andrew C. McNeil, M. Shirin
Sabbaghian, Linda Ehler, Calman Prussin, Randy Stevens,
Laurie Lambert, John Altman, Claire W. Hallahan, Juan
Carlos Lopez Bernaldo de Quiros and Mark Connors
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Copyright © 2000 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2000; 165:1082-1092; ;
doi: 10.4049/jimmunol.165.2.1082
http://www.jimmunol.org/content/165/2/1082
Maintenance of Large Numbers of Virus-Specific CD8ⴙ T
Cells in HIV-Infected Progressors and Long-Term
Nonprogressors
Juan C. Gea-Banacloche,* Stephen A. Migueles,* Lisa Martino,* W. Lesley Shupert,*
Andrew C. McNeil,* M. Shirin Sabbaghian,* Linda Ehler,* Calman Prussin,† Randy Stevens,‡
Laurie Lambert,‡ John Altman,§ Claire W. Hallahan,* Juan Carlos Lopez Bernaldo de Quiros,¶
and Mark Connors1*
A
further understanding of the components, targets, and
magnitude of an effective immune response to HIV are
important steps toward the development of effective prophylactic vaccines or immunotherapies. Induction and maintenance of a primed cellular immune response to HIV may present
an important mechanism of defense that may alter the course of
HIV infection. A number of recent studies have provided direct
evidence for the role of CD8⫹ T cells in restriction of lentiviral
replication in vivo. CD8⫹ T cell depletion by exogenous mAbs has
been shown to abrogate restriction of virus replication in both simian/human immunodeficiency virus-infected or SIV-infected monkeys (1–3). In addition, it has been shown that animals infected
with live attenuated SIV vaccines are able to resist SIV challenge
through Ab- and chemokine-independent mechanisms (4). It now
appears clear that CD8⫹ T cells are an important component to
restriction of virus replication induced by chronic virus infections
in each of these model systems and likely play a similar role in the
restriction of HIV replication in humans. For this reason, considLaboratories of *Immunoregulation and †Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892;
‡
Science Applications International Corp., Frederick Cancer Research and Development Center, Frederick, MD 21702; §Emory University Vaccine Center at Yerkes,
Atlanta, GA 30322; and ¶Servicio de Medicina Interna 1, Clinica Puerta de Hierro,
Universidad Autonoma de Madrid, and Servicio de Microbiologia, Hospital General
Gregorio Maranon, Madrid, Spain
Received for publication November 2, 1999. Accepted for publication April 24, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
Address correspondence and reprint requests to Dr. Mark Connors, National Institutes of Health, Building 10, Room 11B-09, 10 Center Drive, MSC 1876, Besthesda,
MD 20892-1876. E-mail address: [email protected]
Copyright © 2000 by The American Association of Immunologists
erable attention has been focused on the HIV-specific CD8⫹ T cell
responses of patients who are felt to have immune system-mediated restriction of virus replication (5–12). Although patients with
normal CD4⫹ T cell counts and low levels of plasma virus are a
heterogeneous group, a small subgroup of patients with truly nonprogressive HIV infection and restriction of virus replication likely
hold important clues to the basis of an effective immune response
to HIV. However, the targets and magnitude of such responses
necessary for effective restriction of virus replication remain incompletely understood.
A number of techniques have recently become available that
allow the measurement of the magnitude of such responses by
determining the number of Ag-specific CD8⫹ T cells. MHC tetramers permit the determination at the single-cell level of CD8⫹ T
cells specific for a given peptide (13). These reagents have dramatically revised the estimates of the magnitude of the Ag-specific
CD8⫹ T cell response during acute infections of mice and humans
as much as 10- to 100-fold above that previously found by traditional limiting dilution analysis. It has been determined that 40 –
70% of the CD8⫹ T cells during an acute infection of experimental
animals or humans may be Ag specific (14 –17). The total numbers
of Ag-specific CD8⫹ T cells during chronic infections of humans
have not yet been well characterized. MHC tetramer analysis has
recently permitted the quantification of HIV peptide-specific
CD8⫹ T cells in the peripheral blood in some patients with progressive HIV disease (18). However, tetramer analysis alone allows the determination of the number of cells specific for a given
peptide. It is likely the true number of Ag-specific CD8⫹ T cells is
much larger if one were to examine the response to all HIV gene
products in the context of each of the patient’s MHC class I alleles.
Further, although MHC tetramer complexes are powerful reagents
0022-1767/00/$02.00
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The virus-specific CD8ⴙ T cell responses of 21 HIV-infected patients were studied including a unique cohort of long-term nonprogressors with low levels of plasma viral RNA and strong proliferative responses to HIV Ags. HIV-specific CD8ⴙ T cell
responses were studied by a combination of standard cytotoxic T cell (CTL) assays, MHC tetramers, and TCR repertoire analysis.
The frequencies of CD8ⴙ T cells specific to the majority of HIV gene products were measured by flow cytometric detection of
intracellular IFN-␥ in response to HIV-vaccinia recombinant-infected autologous B cells. Very high frequencies (0.8 –18.0%) of
circulating CD8ⴙ T cells were found to be HIV specific. High frequencies of HIV-specific CD8ⴙ T cells were not limited to
long-tern nonprogressors with restriction of plasma virus. No correlation was found between the frequency of HIV-specific CD8ⴙ
T cells and levels of plasma viremia. In each case, the vast majority of cells (up to 17.2%) responded to gag-pol. Repertoire analysis
showed these large numbers of Ag-specific cells were scattered throughout the repertoire and in the majority of cases not contained
within large monoclonal expansions. These data demonstrate that high numbers of HIV-specific CD8ⴙ T cells exist even in patients
with high-level viremia and progressive disease. Further, they suggest that other qualitative parameters of the CD8ⴙ T cell
response may differentiate some patients with very low levels of plasma virus and nonprogressive disease. The Journal of
Immunology, 2000, 165: 1082–1092.
The Journal of Immunology
for examining the frequency of Ag-specific cells, such analyses
provide no direct information on their functional state.
In the present study, we use a combination of assays to examine
the frequency and function of Ag-specific cells in a detailed analysis of 21 HIV-infected patients. Several of these patients are part
of a unique cohort characterized by infection ⬎13 years, normal
CD4⫹ T cell counts, HIV RNA below 50 copies/ml of plasma, and
vigorous HIV-specific proliferative and direct CTL responses.
These patients likely make up ⬍0.8% of HIV-infected individuals
(19 –22). For comparison, we also include patients that fit the more
commonly used clinical definition of nonprogressor and patients
with progressive disease. We analyze the CD8⫹ T cell response by
standard cytotoxic T cell assays, MHC tetramer analysis, and TCR
repertoire analysis. In addition, we have combined techniques of
flow cytometric detection of intracellular IFN-␥ production with
Ag presentation by HIV-vaccinia recombinant-infected autologous
B cells. In this manner, we are better able to determine the global
response to multiple HIV Ags and the functional state of Ag-specific cells.
Patients
Patients 1, 4 – 8, 19 –21, 25, 27, and 29 have not received antiretrovirals
during or before the study period. Patient 3 previously received IFN-␣/
AZT (1/90 to 12/95) or IFN-␣/AZT/DDI (1/96 to 12/96) as part of a National Institute of Allergy and Infectious Diseases protocol. This patient has
remained off of antiretrovirals since that time. Patients 15 and 14 have not
received antiretrovirals in the past 6 mo. HIV infection in study participants
was documented by HIV-1/2 enzyme immunoassay. All subjects signed
informed consent approved by the National Institute of Allergy and Infectious Disease investigational review board. Patients 1– 6 were recently described in a separate report (23). Patients 3– 8, 25, and 101–105 have also
been reported separately (24). The patient numbers remain the same across
studies to permit cross-reference. HLA class I and II typing was performed
by hybridization with sequence-specific oligonucleotide probes following
amplification of the corresponding genes using PCR as described elsewhere (25). CCR5 deletion mutations were detected as previously
described (26).
Cytotoxic T cell assays
PBL were obtained by sodium diatrizoate density centrifugation (OrganonTeknika, Durham, NC) of apheresis donor packs. PBMC were cryopreserved in RPMI 1640 media with 10% FBS and 7.5% DMSO at ⫺140°C.
Standard 51Cr release assays were performed as previously described (27).
Autologous EBV-transformed B cells were infected for 16 h at 37°C with
vaccinia recombinant viruses vVK1 (containing the HIV-1HXB2 gag-pol
gene), vP1287 (HIV-1IIIB gag), vP1289 (HIV-1IIIB p24), vP1290 (HIVIIIB
p17), vP1288 (HIVIIIB pol), vPE16 (HIV-1BH10 env), vTFnef (HIV-1pNL432
nef), or the negative control virus vSC8 (Escherichia coli ␤-galactosidase
(␤-gal)2). Vaccinia recombinants were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program. The
vP1287 (HIV-1IIIB gag), vP1289 (HIV-1IIIB p24), vP1290 (HIVIIIB p17),
and vP1288 (HIVIIIB pol) viruses were contributed to the National Institutes of Health AIDS Research and Reference Reagent Program by Virogenetics (Troy, NY). The vTFnef virus was contributed by MedImmune
(Gaithersburg, MD), and vPE16, vVK1, and VSC8 viruses were contributed by Dr. Bernard Moss (National Institute of Allergy and Infectious
Diseases, Bethesda, MD). Target cells were 51Cr labeled, washed, and
plated at 1 ⫻ 104 cells per well into 96-well round-bottom tissue culture
plates. Fresh or cryopreserved PBMC were used as effectors. Cryopreserved PBMC were cultured overnight at 37°C before use as effectors.
Preliminary experiments have yielded similar results with fresh or cryopreserved PBMC. Effector PBMC were added to each well at 100 –12.5:1.
All CTL assays were performed in triplicate. The percent specific cytotoxicity was calculated as follows: % specific cytotoxicity ⫽ [(experimental
release ⫺ spontaneous release)/(maximal release ⫺ spontaneous release)]
⫻ 100. The lysis of VSC8-infected targets was 12% for patient 5 and ⬍5%
in all other patients in each experiment. Spontaneous release was ⬍20% in
Abbreviations used in this paper: ␤-gal, ␤-galactosidase; TCRBC, TCR ␤-chain
constant region transcript; TCRAC, TCR ␣-chain constant region transcript; bDNA,
branched chain DNA; LTNP, long-term nonprogressor.
2
each experiment. The SE of individual triplicates was 1–20% of mean
specific lysis. In preliminary experiments, significant lysis (⬎10%) was not
observed in MHC class I-mismatched or CD8⫹ T cell-depleted cultures.
All experiments were repeated at least once with similar results.
Flow cytometry
Four-color flow cytometry was performed according to standard protocols
(28). Surface or intracellular staining was performed using the following
Abs: FITC-conjugated anti-IFN-␥ and anti-CD8 (PharMingen, Cupertino,
CA), anti-BV8 and anti-BV5.1 (T Cell Diagnostics, Woburn MA); PEconjugated anti-IFN-␥ (PharMingen), and anti-CD69 (Becton Dickinson,
San Jose, CA) and CyChrome-conjugated anti-CD8 and anti-CD3 (Coulter,
Miami, FL), phycoerythrin-Texas Red-conjugated anti-CD8 and anti-CD3
(Coulter), peridinin chlorophyl protein-conjugated anti-CD3 and anti-CD8
(Becton Dickinson). Cells were analyzed within 24 h using either an EPICS
XL (Beckman Coulter, Fullerton, CA) or a FACScalibur (Becton Dickinson) flow cytometer. Surface staining with anti-BV5.1, anti-BV8, or tetramers was performed following stimulation but before fixation. Then, 0.5
␮l of APC-conjugated HLA-A*0201 tetrameric complex was used to stain
2 ⫻ 106 PBMC in a 50-␮l volume at 4°C for 30 min. MHC class I A*0201
complexed with either of the conserved gag-SLYNTVATL or polILKEPVHGV peptides have been previously described to stain class I-restricted cytotoxic T cells specific for these peptides (18).
Intracellular cytokine detection
Target cells were prepared as described above for the direct CTL assay and
used as the stimulus for intracellular cytokine detection. Intracellular cytokine detection was performed as previously described (29). Briefly, 4 ⫻
106 PBMC were incubated with 400,000 uninfected, vac-␤-gal, or vacHIV-recombinant-infected autologous EBV-transformed B cells in a final
volume of 2 ml of RPMI 1640 containing 10% FBS in 10-ml culture tubes
(Sarstedt, Newton, NC). At 2 h of incubation, brefeldin- A (Sigma, St.
Louis, MO) was added to the medium at a final concentration of 10 ␮g/ml
to inhibit cytokine secretion. At 6 h of incubation, the cells were washed
twice and fixed in 4% paraformaldehyde (Sigma) and permeabilized or
frozen for future use.
Fixed cells were permeabilized and blocked in a solution of PBS with
0.2% saponin (25% sapogenin in content; Sigma), 1 mM CaCl2, 1 mM
MgSO4, 0.05% (w/v) NaN3, 1% BSA, pH 7.4, with 5% nonfat dry milk
overnight at 4°C. Cells were then aliquoted at 1 ⫻ 106 per tube and washed
once in a solution of PBS/saponin. The pellet was resuspended in PBS/
saponin/milk containing Abs for staining and incubated for 30 min at 4°C
in the dark. Samples were then washed twice in PBS/saponin and resuspended in 300 ␮l PBS/BSA 0.1%. Gating was performed on CD3⫹CD8⫹
lymphocytes, and 15,000 –200,000 events (100,000 –700,000 total cells)
were collected. Data were analyzed using either CellQuest (Becton Dickinson) or FlowJo software (TreeStar, Cupertino, CA). Color compensation
settings were made with each round of staining using patient cells labeled
singly with anti-CD3 labeled with specific fluorochromes.
Repertoire analysis
CD4⫹ T cells were isolated by depleting monocytes and CD8⫹ T cells
using anti-CD14- and anti-CD8-coated magnetic beads (Dynal, Lake Success, NY). CD4⫹ T cells were then positively selected with CD4-specific
beads. CD8⫹ T cells were positively selected directly from PBMC with
CD8-specific beads. CD4⫹ or CD8⫹ T cell purity was documented by flow
cytometry to be 95–99%. Total RNA from 107 CD4⫹ or CD8⫹ T cells was
isolated using Trizol LS (Life Technologies, Grand Island, NY) and precipitated with isopropanol in the presence of Microcarrier (Molecular Research Center, Cincinnati, OH). Reverse transcription was performed using
Superscript II (Life Technologies) and oligo(dT) according to manufacturer protocols. Then, 5 ␮g of total RNA was used for analysis of TCRBV
subfamily size patterns, and 1.5 ␮g of total RNA was used for semiquantitative analysis (30).
To analyze TCRBV transcript size patterns, 24 aliquots of the cDNA
were amplified for 40 cycles in 50-␮l reactions. A primer specific for each
of the 22 functional TCRBV subfamilies (BV1-9, BV11-18, BV20-24) (31,
32) and an unlabeled primer specific for the TCR ␤-chain constant region
(TCRBC) was included. The cDNAs were amplified in a Perkin-Elmer
(Foster City, CA) 9600 thermocycler for 40 cycles (denaturation 25 s at
94°C, annealing 45 s at 60°C, extension 45 s at 72°C). Aliquots of the 24
PCR products were then labeled by five cycles of elongation in a “runoff”
reaction with a fluorescent primer (6-carboxyfluorescein-TCRBC) (30, 33).
Products of these reactions were electrophoresed on 24 cm 6% acrylamide
gels on a 373 DNA sequencer and then analyzed using Genescan software
(Perkin-Elmer), as previously described (30).
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Materials and Methods
1083
QUANTIFICATION OF HIV-SPECIFIC CD8⫹ T CELLS
1084
Table I. Clinical data of study patientsa
CD4⫹ T
Cells
(cells/␮l)
CD8⫹ T
Cells
(cells/␮l)
CD8⫹
DR⫹38⫹
(cells/␮l)
Patient
Gender
Age
Plasma HIV RNA
(copies/ml)
Therapy
A
5
7
4
3
6
8
25
M
F
M
M
M
M
M
59
49
49
36
39
47
45
1985
1985
1985
1985
1986
1985
1986
1,105
277
1,063
915
760
664
1,028
835
385
1,088
1,079
803
1,120
1,082
323
104
519
563
528
336
839
⬍50
⬍50
⬍50
⬍50
⬍50
⬍50–324
⬍50–1,089
–
–
–
–
–
–
–
B
1
20
21
19
M
M
M
M
38
42
52
46
1985
1985
1984
1984
693
1,311
725
785
630
1,001
834
2,319
488
320
225
1,136
3,996
4,825
4,826
7,774
–
–
–
–
C
27
15
29
14
F
M
M
M
28
27
24
29
1993
1996
1999
1998
170
255
488
410
634
637
886
1,087
NTb
223
NT
554
2,486
6,655
36,475
53,158
–
–
–
–
D
2
105
104
102
101
103
M
M
M
M
M
M
53
38
40
34
41
50
1986
1990
1988
1993
1986
1991
724
635
271
767
483
618
1,930
784
746
1,505
877
894
1,176
196
319
497
307
215
⬍500–14,080
1,008
1,702
2,940
7,817
7,873
AZT
D4T/DDC/NEL
3TC/D4T/IND
D4T/DDC/IND
AZT/3TC/NEL/IL-2
3TC/D4T/IND/IL-2
a
Mean CD4⫹ and CD8⫹ T cell counts of uninfected individuals are 912 ⫾ 24.08 and 528 ⫾ 20.8 cells/mm2, respectively, in this laboratory. Determination of plasma virus
was performed using bDNA assay (Chiron) with a 50-copies/ml plasma sensitivity. In patients 1 and 2, plasma virus was measured using an earlier version of this assay with
a 500-copies/ml sensitivity.
b
NT, not tested.
To quantify TCRBV transcripts, 24 aliquots of the cDNA were amplified in 50-␮l reactions as above except the primer specific for the TCRBC
was labeled with the blue fluorescent label 6-carboxyfluorescein (34), and
a primer set specific for a TCR ␣-chain constant region transcript (TCRAC) was added to provide an internal control (35). The 3⬘ TCRAC primer
was labeled with a green fluorescent label tetrachloro-6-carboxyfluorescein
(TET). Thirty cycles of PCR were used. The products were electrophoresed
and analyzed for size and fluorescence intensity as previously described
(30, 36).
Sequence analysis
The BV subfamily-specific primers used in semiquantitative and size pattern analysis were modified to contain an additional 12 base sequences
containing uracil for direct cloning into the pAMP vector (Life Technologies) according to the manufacturer’s protocol. PCR amplification from
cDNA was conducted as described above for 40 cycles, and 5 ␮l of this
reaction was used to anneal into the vector. The product was then used to
transform DH5⬀ competent cells (Life Technologies). DNA was isolated
using plasmid isolation kit (Qiagen, Chatsworth, CA) and sequenced using
FS dye terminator cycle sequencing (Perkin-Elmer) and electrophoresed on
6% polyacrylamide gels in a Perkin-Elmer 373 automated sequencer.
Results
5,997, 10,045, and 2,911, respectively) when stimulated with 8
␮g/ml of p24 Ag (A. McNeil, unpublished observations). These
responses are similar to those recently described in two patients
with plasma viral RNA below 50 copies/ml plasma (11, 37). In all
other patients in the present study, the proliferative response to p24
was ⬍1000 ⌬cpm, which was equivalent to uninfected controls.
Patients within group B meet more commonly used clinical criteria
of nonprogressive disease (infection ⬎7 years, peripheral CD4⫹ T
cell count ⬎500 cells/␮l without antiretroviral use) (38, 39).
Group C contains patients with progressive disease not receiving
antiretrovirals. Because of the lack of availability of patients with
progressive disease not receiving antiretroviral therapy, six patients (group D) receiving therapy but with plasma virus RNA
levels ⬎1000 copies/ml were included for comparison. The MHC
class I and class II haplotypes of the patients are shown in Table
II. HLA B*57 was overrepresented in group A patients that are
part of a larger cohort of such patients examined in further detail
in another report (24). Only patient 25 was heterozygous for the
32-bp deletion within the HIV coreceptor CCR5.
Patient characteristics
The clinical characteristics of the study patients are shown on Table I. For the purpose of discussion patients were divided into four
groups. Patients within group A have been infected with HIV for
at least 13 years, with stable peripheral blood CD4⫹ T cell counts
between 690 and 1200 cells/mm3 and typically maintain plasma
virus levels below 50 copies/ml measured by branched chain DNA
(bDNA). Patient 8 has not had viremia detected by bDNA since
diagnosis until a recent increase to 324 copies/ml associated with
a febrile illness. Patients 3– 6 have shown strong proliferative responses to p24 Ag (⌬cpm 9,752–27,778; stimulation index 10 –
140) in conventional lymphoproliferation assays (23). Using the
same assay, similar CD4⫹ T cell-mediated proliferative responses
to HIV Ags have also been found in patients 7, 8, and 25 (⌬cpm
Direct CTL activity
The magnitude of HIV-specific CD8⫹ T cell responses detected by
direct CTL assays in a subset of patients was determined. The
results are shown in Fig. 1. In preliminary experiments, specific
lysis above 10% was not reproducibly observed in uninfected individuals. Positive CTL activity was considered to be ⬎10% specific lysis. In preliminary experiments, differences in results obtained with fresh or cryopreserved samples varied ⬍10% at a
given E:T ratio, which is within the variability of this assay. These
assays were performed without restimulation of effectors and in the
absence of exogenous IL-2. The predominant activity was against
gag-pol (10 –30% sp. act.), followed by nef and by env. The level
of this activity is consistent with that previously published for
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Group
Year of
Diagnosis
The Journal of Immunology
1085
Table II. Patient HLA class I and II alleles
MHC Class I
MHC Class II
Patient
A*
B*
C*
DRB1*
DQB1*
DRB
A
5
7
4
3
6
8
25
2, 24
1, 2
1, 31
2, 3
11, 30
11, 23
3, 24
57
57
8, 57
13, 39
52, 57
44, 57
27, 57
6, 7
6
6, 7
6, 7
7, 12
4, 6
2, 6
7, 13
7, 14
3, 13
7, 16
13, 15
7, 11
4, 8
3, 6
3, 5
2, 6
2, 3
6
2, 3
3, 4
3, 4
3, 4
3
4, 5
3, 5
3, 4
4
B
1
20
21
19
3, 66
1
1, 2
2, 3
7, 58
52, 57
8, 27
14, 44
7
6, 12
1, 7
5, 8
11, 15
13, 15
1, 3
4, 13
3, 6
3, 6
2, 3
3, 6
3, 5
3, 5
3
3, 4
C
27
15
29
14
2, 36
68
29
36, 68
15, 42
15, 58
44, 49
45, 57
3, 17
3, 6
7
7, 16
3
8, 12
1, 7
3, 11
2, 4
3, 5
2, 5
4, 5
3
3
4
3
D
2
105
104
102
101
103
2
2, 80
2
24, 68
1, 31
2, 11
40, 51
8, 57
57, 58
15, 57
51, 57
55, 57
3, 14
2, 7
3, 6
6, 7
6, 15
3, 6
4, 7
3
7, 11
1, 7
7, 15
7
2, 3
2
3
3, 5
3, 6
3
4
3
3, 4
4
4, 5
4
patients with progressive or nonprogressive disease (Ref. 40; reviewed in Ref. 41).
HIV Ag-specific IFN-␥ secretion
To further characterize the CD8⫹ T cell response to HIV Ags by
a more quantitative method, we adapted a method of intracellular
cytokine staining detected by flow cytometry to enumerate Agspecific CD8⫹ T cells at the single cell level. HIV-vaccinia recombinant-infected EBV-transformed autologous B cells were
used as stimuli. Similar assays have been used with peptide-pulsed
target cells to measure responses to CMV in one report in humans
and in virus infections in mice (16, 17, 42). Such assays have the
advantage of not requiring in vitro proliferation of effector CD8⫹
T cells, which likely results in the low and variable numbers of
these cells detected by traditional limiting dilution analysis. In addition, the determination of the response to the products of whole
genes in the present study is not limited to a single peptide and
permits a determination of the global response to HIV Ags.
An example of intracellular staining of stimulated cells from
patient 8 is shown in Fig. 2A. Gating on CD3⫹ CD8bright lymphocytes, the percent of cells that were CD69⫹ IFN-␥⫹ in response to
a given stimulus was determined. No CD3⫹CD8⫹ cells were
shown to synthesize IFN-␥ after a 6-h incubation in the absence of
stimulation. When PBMC were incubated with uninfected or vac␤-gal-infected EBV-transformed autologous B cells, the background percent positive cells was 6.87 for patient 5 and more typically between 0.04 and 2 for the remaining patients. As the
percents were equivalent under these two conditions, it is likely
FIGURE 1. Direct cytolysis of autologous HIV-vaccinia-recombinant-infected autologous EBV-transformed B cells. Specific lysis shown is after
subtraction of lysis of vsc-8(␤-gal)-infected cells.
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Group
1086
QUANTIFICATION OF HIV-SPECIFIC CD8⫹ T CELLS
these cells were activated by either the B cells alone or EBV Ags
(43) and are not vaccinia specific. The response to individual gene
products is shown in Fig. 2B. The sum of the responses to gag
and pol is equivalent to the response to the gag-pol construct
gene products. Similarly, the sum of responses to p17 and p24
is equivalent to the gag response, indicating this method is
highly quantitative. Similarly, the percent of CD8⫹ T cells that
produce IFN-␥ in response to peptide-pulsed B cells closely correlates with the percent obtained by the corresponding peptide
MHC tetramers (not shown), consistent with recent results obtained by an IFN-␥ enzyme-linked immunospot assay (44). The
observed frequencies of Ag-specific cells were also highly reproducible by this method in repeated experiments in each of the
patients studied.
The summary data of the percent of CD3⫹CD8⫹ lymphocytes
which were CD69⫹ IFN-␥⫹ in response to a given HIV gene product are shown in Fig. 3. By this method, the fraction of cells that
were HIV specific varied from 0.8 to 18.0%. Preliminary experiments over a broad range of E:T ratios showed no higher response
to increased target numbers, only higher background activity to the
vac-␤-gal control. Similar to the direct CTL data, the predominant
activity was again directed against gag-pol with a frequency of
CD69⫹ IFN-␥⫹ cells between 0.43 and 17.02%. The response in
patient 8 was observed in multiple samples taken over several
months and is dramatically higher than that observed in other patients. It is also higher than one would predict based upon direct
CTL. It is possible this response is related to a recent increase in
plasma viremia associated with a febrile illness and may diminish
with time. With the exception of the response in patient 8, there
was a good correlation between the percent of CD8⫹ T cells reacting to gag-pol and direct cytolytic activity to this target (R2 ⫽
0.796, p ⫽ 0.007). If the response of patient 8 is included in this
correlation, it is no longer significant (R2 ⫽ 0.3, p ⫽ 0.15). The
FIGURE 3. The percent CD3⫹ CD8⫹ cells that are CD69⫹ IFN-␥⫹ in
response to autologous B cells infected with HIV-vaccinia recombinants
encoding the indicated gene product. The percents shown are with the
background ␤-gal activity subtracted.
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FIGURE 2. A, IFN-␥ production in CD8⫹
T cells in unstimulated PBMC or those stimulated with ␤-gal control or the indicated HIV
gene product. PBMC were incubated with autologous-transformed B cells infected with
vaccinia-HIV recombinants at an E:T of 10:1.
PBMC were fixed, permeabilized, and stained
following a 6-h stimulation. Gating on
CD3⫹CD8⫹ cells the plots depict surface
staining for the activation marker CD69 and
intracellular IFN-␥. B, The percent of
CD3⫹CD8⫹ cells that are CD69⫹ IFN-␥⫹ in
response to the indicated gene product for patient 8. The percents shown are with the background ␤-gal activity subtracted.
The Journal of Immunology
1087
lack of a single-cell assay for cytolytic activity does not permit the
effector CTL function of IFN-␥-producing cells to be directly confirmed. It has recently been shown in humans and in mouse models
of viral infection that even memory CD8⫹ T cells will rapidly
secrete IFN-␥ upon restimulation and may remain cytolytically
active (16, 45, 46). Thus, the cells detected in the current study by
intracellular cytokine staining are likely a composite of Ag-experienced cells with either memory or effector function.
High numbers of Ag-specific CD8⫹ T cells were not unique to
long-term nonprogressor (LTNP) patients with low levels of
plasma viral RNA and strong proliferative responses. In some
cases, the lowest frequencies were observed in LTNP (patients
such as 5 and 6) with ⬍50 copies of viral RNA/ml plasma. No
significant difference in the number of HIV-specific CD8⫹ T cells
were observed in group A (mean 7.7 ⫾ 2.1) compared with all
other patients not receiving antiretrovirals (mean 7.5 ⫾ 2.1, p ⫽
0.5), even those with progressive disease and high-level viremia.
No higher frequency of gag-specific CD8⫹ T cells was found in
group A when compared with other patients not receiving antiretrovirals ( p ⫽ 0.3). No significant correlation between the number
of HIV-specific CD8⫹ T cells and the level of plasma viral RNA
was found ( p ⫽ 0.3). Because of the lack of availability of patients
with progressive disease not receiving antiretroviral therapy, six
patients with progressive disease on therapy with ⬎1000 copies of
plasma viral RNA were included (group D). Again, large numbers
of HIV-specific cells were observed, and no significant difference
was found when compared with group A (mean 4.4 ⫾ 1.2, p ⫽
0.5). Given that antiretroviral therapy decreases the number of
HIV-specific CD8⫹ T cells (47), these results likely underestimate
the total number of Ag-specific cells that would be observed in
these patients if therapy was removed. These results do indicate
that the number of HIV-specific CD8⫹ T cells is no lower in patients with progressive disease when compared with nonprogressors with high-level restriction of plasma viremia.
In some virus infections in experimental animals, the number of
IFN-␥-secreting CD8⫹ T cells specific for a given peptide has been
found to be similar to that detected by MHC tetramers (16, 17).
Given that such large numbers of Ag-specific cells were found in
HIV-infected patients, one concern is that the number of activated
cells detected may be increased through bystander effects of cytokine secretion in vitro. However, the duration (6 h) of the assay
and addition of brefeldin-A to stop cytokine secretion makes this
possibility unlikely. To address these issues, we used two wellcharacterized HLA-A*0201 tetramer complexes to detect Ag-specific cells and determine the fraction able to secrete IFN-␥ under
the current experimental conditions (18).
Four of 11 patients tested were MHC class I A*0201 positive.
Of these patients, only two had ⬎0.1% of CD8⫹ T cells specific
for the conserved gag (SLYNTVATL) or pol (ILKEPVHGV) peptide. The tetramer staining of PBMC of patients 3 and 19 is shown
in Fig. 4A. No bystander activation of these cells was observed
when cells were stimulated with any recombinant encoding nonp17 gene products. Although no bystander activation was observed, many MHC tetramer⫹ cells did not produce IFN-␥. Under
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FIGURE 4. A, IFN-␥ production of CD8⫹ T cells of patients 3 and 19 that stain with MHC-gag tetramer complex. Cells were prepared as in Fig. 2A.
Additionally, cells were stained with the HLA A2 (SLYNTVATL) peptide complex. B, Response of CD8⫹ T cells with increased numbers of vacciniagag-infected target cells (E:T ratio is 1:1) or PMA/ionomycin.
1088
QUANTIFICATION OF HIV-SPECIFIC CD8⫹ T CELLS
the experimental conditions in Fig. 4A, almost one-half of MHC
tetramer⫹ cells did not produce IFN-␥ upon stimulation. Because
the frequency of SLYNTVATL-specific cells is lower than the
total responsive to the entire gag-pol gene product (Fig. 3), the
number of target cells of patient 3 was increased to a 1:1 ratio to
engage all Ag-specific cells (Fig. 4B). This increases the numbers
of cells responding but also dramatically increases the nonspecific
activity to other Ags such as to ␤-gal. Under these conditions, 73%
of MHC tetramer⫹ cells could be induced to make IFN-␥. Higher
numbers of responding MHC tetramer⫹ cells were not observed
with higher numbers of target cells or longer stimulation at 12 or
24 h. Thus, of all MHC-gag tetramer⫹ cells only a subset produced
IFN-␥ when stimulated with the gag or gag-pol gene product.
Nearly all MHC-gag tetramer⫹ cells of patients 3 or 19 were ultimately capable of producing detectable IFN-␥ after stimulation
with PMA/ionomycin (Fig. 3B). Surface staining was used to further characterize the population of CD3⫹CD8⫹ MHC tetramer⫹
cells. Of unstimulated CD3⫹CD8⫹ MHC tetramer⫹ cells, 46%
were CD38⫹, 90% were CD27⫹, and 72% were CD45RA⫺. This
result is consistent with the vast majority of these cells being Agexperienced memory or effector cells (13, 18, 48).
Analysis of the CD8⫹ T cell receptor repertoire
Large virus-specific expansions previously have been found in
some acute and chronic infections in humans (15, 43, 49 –51).
Given that patients in the present study had large numbers of Agspecific cells, it was of interest to determine whether large HIV-
specific expansions existed within the CD8⫹ T cell Ag receptor
repertoire. Of the patients examined (1– 6), large expansions
within the CD8⫹ TCR repertoire were found in patients 3– 6. In
each case, these expansions were associated with expansion of a
single-sized transcript (Fig. 5). Sequence analysis revealed that
these expansions were monoclonal. In one case (patient 3), this
monoclonal expansion made up ⬃30% of the circulating CD8⫹ T
cells. In patients 3 and 6, these expansions were shown to persist
over 4 years of study. The CD4⫹ T cell repertoire was also analyzed in these patients and found to be polyclonal. No subfamily
was expanded to ⬎15%, and all size pattern distributions were
gaussian and indistinguishable from those of uninfected
individuals.
We then determined if HIV specificity of these expanded CD8⫹
T cell clones could be detected (Fig. 6). BV subfamily-specific
Abs were used to stain the CD8⫹ T cell expansions of patients 3–5.
No BV6 Ab that recognized the expansion of patient 6 was found.
The percent of CD8⫹ T cells that stained with BV-specific Ab
correlated well with the percentages detected by semiquantitative
PCR. No HIV specificity could be found within the BV8 expansion
of patient 3 and the BV5 expansion of patient 5. Although HIV
specificity was not demonstrated for most of the expansions found,
the BV5.1 expansion of patient 4 was found to be nef specific.
Some decrease in the intensity of BV5.1 Ab staining is observed
on IFN-␥⫹ cells, consistent with activation-induced TCR downregulation. Small numbers of gag-pol-specific cells were also detected in this subfamily and likely represent the lower frequency
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FIGURE 5. CD8⫹ T cell repertoire of patients 3– 6. CD8⫹ T cells were isolated from PBMC in high purity and RT-PCR amplified from mRNA.
Semiquantitative PCR, size pattern analysis, and sequence analysis for the indicated BV subfamilies is shown.
The Journal of Immunology
1089
clones detected by size pattern analysis and sequencing of BV5.1
in this patient. Although the expansion makes up the vast majority
of clones in the BV5.1 subfamily, only 20% of the BV5.1⫹ cells
produced IFN-␥ in response to the nef gene product. Higher numbers of IFN-␥-producing cells were not observed in response to
higher numbers of infected target cells.
Discussion
In the present study, very high numbers of HIV-specific CD8⫹ T
cells were detected in patients across a broad spectrum of plasma
virus load. Using a method that couples the capability of studying
the frequency of Ag-specific cells provided by intracellular cytokine staining with the ability to study a wide array of epitopes
inherent to the HIV-vaccinia recombinant-infected B cell system,
between 0.8% and 18% of the circulating CD8⫹ T cells were
found to be specific for HIV. These numbers are ⬃3–15 times the
number previously estimated by enzyme-linked immunospot or
tetramer analysis (18, 40, 47, 52–54). In some cases, this percent
approaches the total number of CD3⫹CD38⫹ T cells previously
believed to be mostly due to bystander activation in infected patients (21). This study includes a unique cohort of LTNPs that has
been infected ⬎13 years, has strong proliferative responses to HIV
Ags, and maintains plasma virus levels ⬍50 copies/ml. Although
these patients appear to have a potent ability to restrict virus replication, this was not associated with higher numbers of HIV-specific CD8⫹ T cells measured in vitro. Similar high levels of virusspecific CD8⫹ T cells were observed in these patients as some
patients with progressive infection and 55,000 copies of viral RNA
per ml of plasma or other patients with plasma viremia poorly
controlled by antiretroviral therapy. It has previously been proposed that patients that maintain effective restriction of HIV replication do so because of greater HIV-specific CD8⫹ T cell responses. However, previous studies have not consistently
demonstrated a significant correlation between levels of plasma
virus and CD8⫹ T cell responses measured in vitro (7, 8, 10, 21,
37, 55, 56). It has been further suggested that significant correlations between plasma virus levels and measures of HIV-specific
CD8⫹ T cell responses were not detected because the assays used
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FIGURE 6. HIV specificity of CD8⫹ T cell BV subfamily expansions. See Fig. 2A. CD3⫹CD8⫹ cells that stained with the indicated BV subfamily
subtype-specific Ab and produce IFN-␥ in response to the indicated stimuli are shown.
1090
cells make IFN-␥ in response to HIV p55 Ag (63). Taken together,
these data are then consistent with those from experimental animals showing that the CD4⫹ T cell repertoire is much less prone
to large monoclonal expansions and the numbers of virus-specific
CD8⫹ T cells are as much as 10-fold larger than those of CD4⫹ T
cells (49, 61, 64 – 66).
Although large numbers of Ag-specific cells were in some cases
detected by either tetramer analysis or repertoire analysis, only a
subset of these cells were able to activate, as measured by CD69
staining, or produce IFN-␥ when stimulated through the TCR.
Only approximately one-half of the MHC tetramer⫹ cells of patients 3 or 19 produced IFN-␥ in response to the appropriate HIV
gene product. It is possible that the MHC tetramer⫹ IFN-␥⫺ cells
are unable to make IFN-␥ after TCR engagement such as CCR7⫹
memory cells (67) or alternatively noneffector Ag-specific cells
recently observed (68). However, unlike the situation in lymphocytic choriomeningitis virus-infected mice under conditions of
CD4⫹ T cell depletion, tetramer⫹ IFN-␥⫺ cells in the present
study were able to produce cytokine upon stimulation with PMA/
ionomycin. It is also possible that these MHC tetramer⫹ IFN-␥⫺
cells are a subpopulation of MHC tetramer⫹ cells that are not Ag
specific. This is a possibility given the avidity of the MHC-peptide
complex was increased by producing tetramers to allow staining of
Ag-specific cells. However, this is not necessarily the case given
the BV 5.1 expansion of patient 5 was nef specific and monoclonal
yet only 20% of these cells produced IFN-␥ even under conditions
of high numbers of infected APCs. This result suggests that the
Ag-specific cells detected by MHC tetramers that do not activate
or accumulate cytokine in response to HIV gene products may be
Ag-specific cells of relatively low avidity and may have a more
limited ability to activate in response to stimulation through
the TCR.
It should be noted that conclusions regarding protective immunity based upon correlations between plasma viremia and parameters of HIV-specific immunity should be approached with some
caution. Because no inverse relationship was found between
plasma virus and the numbers of Ag-specific CD8⫹ T cells measured in vitro does not imply these cells are not important mediators of protective immunity or restriction of virus replication in
some infected patients. For reasons pointed out above the relationship between the measured CD8⫹ T cell response and plasma viremia is likely quite complex and dependent upon virus replication
and stage of disease. Further, the qualitative nature of the CD8⫹ T
cell response may be quite different in the context of a vaccine that
might induce CD4⫹ and CD8⫹ T cell responses than in HIVinfected individuals in the context of diminished CD4⫹ T cell help.
It is now clear by more direct evidence that CD8⫹ T cells are
important mediators of the restriction of virus replication observed
in several experimental animal models of HIV infection (1– 4).
Similarly, the cells of patients 3– 8 are able to restrict autologous
and challenge virus replication when engrafted into SCID-Hu animals and restriction of challenge virus replication is abrogated by
CD8⫹ T cell depletion (23). However, this activity was not correlated with higher CD8⫹ T cell responses in standard assays of
suppression or cytolysis. Similarly, such patients do not appear to
be distinguished by higher numbers of Ag-specific CD8⫹ T cells
than patients with progressive disease in the present study.
Although high numbers of HIV Ag-specific CD8⫹ T cells are
maintained in infected individuals with progressive disease, these
appear to have a limited capacity to restrict virus replication. The
important question that remains from these results and those of
others is how high-level viremia persists in many patients despite
such large numbers of HIV-specific CD8⫹ T cells. It appears that
the ability of patients within group A to restrict virus replication
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require in vitro stimulation, are poorly reproducible, or are not
reliably quantitative. Subsequent more quantitative analyses using
MHC tetramers dramatically increased estimates of the numbers of
HIV-specific CD8⫹ T cells by 10- to 100-fold and demonstrated an
inverse correlation with plasma viremia (18). In contrast, a similar
correlation was not observed in the present study. However, these
results are in agreement with one very recent report in which the
numbers of CD8⫹ T cells specific for previously described
HLA-A- and -B-restricted peptides detected by enzyme-linked
immunospot assays in patients with progressive HIV infection did
not correlate with plasma viral load or CD4⫹ T cell count (54).
There are a number of important differences that may account
for the apparent inconsistencies between the results of the present
study with recent observations using HIV peptide MHC tetramers
(18). First, the patient population studied here is shifted toward
patients with nonprogressive infection with lower plasma virus
loads. Previous studies have identified patients with low levels of
HIV-specific CD8⫹ T cell responses in end stage disease (8, 54).
Conversely, low responses have also been observed by a variety of
methods in some LTNPs with very low virus loads similar to patients 5 and 6 (8, 21, 40, 54) suggesting the measured CD8⫹ T cell
responses may be dependent on virus replication in such patients.
Thus, the results of such correlations might be dramatically affected by inclusion of patients at either extreme of HIV infection.
Second, the number of Ag-specific cells even for conserved peptide sequences is highly variable across patients regardless of viral
burden. Although four of the patients tested are A2 positive, only
patients 3 and 19 stain ⬎0.1% of CD8⫹ T cells with the SLYN
TVATL-A2 tetramer, consistent with some previous observations
(40, 53). Further, upon mapping the response to gag peptides of
nine of these patients, the number of IFN-␥⫹ cells detected in
response to a given peptide restricted to a single MHC allele may
range from 0 to 5% with no association with viral burden (24, 54).
Last, tetramer analysis alone examines the response specific for a
given MHC allele. Because of the difficulties of mapping and production of tetramers of a given peptide, analysis is commonly done
on only the more common MHC A and B alleles. Thus, when the
global response to HIV gene products in the context of each of a
given patient’s MHC alleles is measured, considerably higher frequencies of Ag-specific cells are found. Although these frequencies are quite high, it is likely they still may underestimate the true
frequency of HIV-specific cells if one were to measure the total
response to the patient’s virus or cells in the lymphoid tissues.
Although large percentages of the circulating cells were specific
for HIV they were not typically concentrated within large monoclonal expansions but rather were scattered throughout the TCR
repertoire. Overall, this is consistent with one recent report in
which gag tetramer⫹ cells were found to be contained within some
expanded BV subfamilies by flow cytometry (52). Upon examination of the CD8⫹ TCR repertoire of the patients in the present
study, some extremely large monoclonal expansions (up to 30%)
were observed. Yet in only a minority of cases were these expansions found to be specific for the HIV isolate tested. It is possible
these expansions are in fact HIV specific and do not react with the
isolates represented by the vaccinia recombinants used. Alternatively, they are not HIV specific and similar to those observed in
HIV-uninfected individuals (57– 62) or are specific for other
chronic virus infections such as CMV or EBV Ags not actively
expressed in transformed B cells. The lack of similar expansions
within the CD4⫹ T cell compartment confirms the observed expansions in CD8⫹ T cells are not due to superantigen effects nor
contamination with CD4⫹ T cells. An analysis of Ag-specific
CD4⫹ T cells in some patients in the present study has shown that
in these patients between 0.2% and 0.8% of circulating CD4⫹ T
QUANTIFICATION OF HIV-SPECIFIC CD8⫹ T CELLS
The Journal of Immunology
may lie not in the number of virus-specific cells but likely in other,
qualitative measures of their CD8⫹ T cell response that are not
accounted for in traditional assays. It should be mentioned that in
several models of anti-tumor responses or disruption of CD4⫹ T
cell function during virus infection, tumor or infection is poorly
controlled by CD8⫹ T cells in vivo yet may retain cytolytic activity detected in vitro (69 –74). In addition to CD8⫹ T cell avidity,
other measures of the peptide targets, CD8⫹ T cell-derived suppressive factors, and MHC down-regulation, which may better
model in vivo restriction of virus replication, are each being pursued as part of ongoing work. Further studies of such patients with
very low levels of plasma virus and maintenance of strong proliferative responses may provide important clues to qualitative differences in the HIV-specific immune response that lead to effective
restriction of virus replication.
1091
16.
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19.
20.
21.
Acknowledgments
22.
23.
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QUANTIFICATION OF HIV-SPECIFIC CD8⫹ T CELLS